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Genomics and Proteomics
March 1, 2004SECTION:
Features; Genomics; Pg. 26LENGTH:
1854 wordsHEADLINE: Helping SNPs to Speak Up
Louder Than Before;
Studying genetic variation generally shows
much promise, but finding the phase or haplotype of a set of SNPs is tricky
business. Experimental approaches are being explored that could facilitate
haplotype analysis for SNPs separated by longer stretchesBYLINE:
By Vivien MarxBODY:
If single-nucleotide polymorphisms (SNPs) offer quiet evidence of
genetic diversity, then haplotypes crank up the volume. Researchers are working
on better methods to let them hear the precise tune that haplotype blocks of
genomic variation play. Single-molecule techniques, for example, permit a new
opportunity for long-range haplotyping. Some techniques being explored involve
the co-amplification of two loci on the same molecule, i.e., two 100-base-pair
loci rather than one 20-kilobase (kb) stretch that has both SNPs on it.
In a given cell, haplotyping, in particular resolving the
phases of several markers, is a challenge. If a region has three heterozygous
markers, that means eight possible haplotypes. Pedigree information and
knowledge of computational mechanisms can help hone in on those possibilities.
Other methods that do not require pedigree information involve haplotyping the
direct molecular way, by separating the homologous DNAs before genotyping. Then
comes DNA cloning, hybrid construction, and single-cell long-range PCR, all of
which are time-consuming and limited to short genomic stretches. Computational
methods, such as inference from unphased data, have limitations, for example,
when it comes to genomic length.
It all depends on the
locus, but computational inference is a hard problem, says Robi Mitra, PhD,
assistant professor of Genetics at Washington University School of Medicine, St.
Louis. "In some haplotypes," he says, "just by eye you see where recombination
events occur, that's a no-brainer." And then there are areas where heterogeneity
and the number of haplotypes, the recombination, all make the task
computationally hard with plenty of probabilistic solutions given the data.
The problem with statistical and computational methods,
says Charles Cantor, PhD, chief scientific officer of Sequenom Inc., San Diego,
is their mistakes. "If you are going to do haplotyping as part of a population
analysis," he says, "you may be tolerant of a certain percentage of mistakes,
but if you are going to haplotype in a clinical diagnostic setting, you are not
tolerant to any those kinds of things."
currently being explored in a number of labs is haplotyping with single-molecule
amplification. One method, molecular colony technique, developed in Alexander
Chetverin's lab at the Institute of Protein Research of the Russian Academy of
Sciences involves amplification in acrylamide. There is BEAM-ing, a word derived
from the method's components of beads, emulsion, amplification, and magnetics.
Developed by Devin Dressman, PhD, and colleagues in Bert Vogelstein's lab at
Johns Hopkins, it involves converting each DNA molecule into a single magnetic
particle to which thousands of identical copies of DNA are bound.
Polony: a sausage and a colony
Another method is called polymerase colony amplification or "polony"
technology for short. Mitra started working on polonies as a graduate student in
George Church's lab at the Lipper Center for Computational Genetics in the
Department of Genetics at Harvard Medical School. It was Church's idea to
amplify single molecules in acrylamide and they subsequently worked out
different applications such as sequencing and haplotyping.
"This is a way to amplify a large number of single DNA molecules," says
Mitra. Much like the apples and the proverbial tree, due to the constraints of
the acrylamide, each of the amplification products remains localized near its
parent molecule. Every molecule in the colony has originated from the same
parent molecule, so large numbers of PCR reactions can happen at the same time
while keeping the resulting DNA separate.
these techniques represent the beginning stages, interviewed scientists point
out they all show the power of analyzing single molecules rather than
populations of molecules.
To illustrate polony
haplotyping, Mitra, Church and colleagues performed an in-gel PCR reaction with
a small amount of patient DNA with two pairs of PCR primers capable of
amplifying one of the SNPs. Thus, two loci from a single DNA molecule are
amplified. The gel's structure keeps the amplification products close by the
chromosome as two overlapping polonies. One strand of the amplified DNA is
attached to the matrix via a covalent bond permitting removal of the other one.
The polonies were genotyped and the phase of the SNPs determined by the
overlapping polonies. Locus-specific primers carrying fluorescent dyes helped to
The team managed to determine the
genotype and phase of three different pairs of SNPs and, in one case, the SNPs
were at a distance of 45 kb. The distance, says Mitra, is because they used DNA
prepared by standard methods, which tends to be sheared at about 50 kb sizes.
The distance limit is due to their DNA source, he says. "Basically," Mitra says,
"there is no reason that with this technique we can't go longer with just a
different purification method."
Another shot at
Cantor, along with Chunming Ding, PhD,
of the Bioinformatics Program at the Center for Advanced Biotechnology, Boston
University, proposes M1-PCR as their road to more success for direct molecular
haplotyping of long-range genomic DNA.
explains, very little DNA is needed, "only 3 pg, which is 1,000 times less than
for a typical genotyping assay." No DNA from parents or siblings is required and
the assay works on DNAs extracted from peripheral blood, thus the time consuming
steps of cell line construction or DNA cloning are avoided.
In M1-PCR, the "M" stands for multiplexing and the "1" for single-copy
DNA molecules. The process entails taking a DNA sample and diluting it to
single-copy, which separates the two homologous DNAs. Then comes the direct
multiplex genotyping of several markers with Sequenom's MALDI-TOF MS-based
MassArray system. A built-in software tool automatically categorizes each
analyzed sample as to whether one or both alleles are present.
Applying this system, the scientists were able to haplotype several
polymorphic markers separated by as many as 24 kb. "We can multiplex and so when
we start with a single molecule, we do a multiplex of short PCRs flanking the
particular SNP," says Cantor. "We amplify just around the SNP instead of doing
the long stretch." Only approximately 100 base pairs of the region around each
SNP are amplified through PCR in this process. By integrating M1-PCR into the
MALDI-TOF system, the scientists say that high-throughput direct molecular
haplotyping of a few thousand assays a day can be obtained.
As part of their experiments, Ding and Cantor compared the haplotypes
determined by their method with those determined through genotyping and with
pedigree information. At first, their method yielded a number of incomplete
haplotype calls, which stood out. "When there are breaks in the molecule, what
we see is an incomplete genotype or haplotype, Cantor says. "And then we know
that our single molecule was missing an end."
trick, says Cantor, is to have enough replicates. In order to counter this
effect, they used more replicates, 10 to 14 replicates, of single-molecule PCR
for each individual. "This is not a problem, since most of the cost of doing
this is in the sample preparation," he says. "Once you are diluting the sample
to one molecule, the cost is insignificant." Distance between SNPs is not a
limiting factor, the researchers say, as long as not many copies of the genomic
DNA have breaks between the markers.
Cantor and Ding
say that their technique is applicable to clinical settings where speed, sample
size, and ease of use are all key. They realize it must be validated on lots of
samples. "We have to make sure that an average lab can do it, not just a lab
with our experience," Cantor says.
There was a
Back in the 1980s, Gualberto Ruano, MD, PhD,
former CEO of Genaissance Pharmaceuticals, who founded Genomas LLC, both of New
Haven, Conn., explored the single-molecule dilution, but found he could not get
two or three amplifications at the same time. "We always knew the limit of this
method would be imposed by the structure of the DNA molecule," he says.
In his 1990 paper, he outlined how haplotypes could be
applied, much of which has come to pass. "I am a good prophet," Ruano says. "It
is extremely satisfying to see the technique advancing." Part of that push was
to found Genaissance Pharmaceuticals Inc., to integrate gene variation into the
development of drugs. He does regret that he was "too naive" to patent his
methods. "Then again, the patents would be close to expiring now," he says.
Into the clinic, slowly
Collecting families for analysis is time-consuming, Cantor points out,
and may be unrealistic for a clinical diagnostic setting in which rapid
decisions are needed. Chunming Ding offers a possible scenario: "For example,
the R117H mutation in the cystic fibrosis transmembrane receptor (CFTR) gene
shows mild effect with the 5T mutation, and severe effect when the 5T mutation
is present on the same chromosome. Thus, a haplotype of R117H-5T is important
for clinical applications to determine the severity of the prognosis of this
type of cystic fibrosis." There are other diseases in which a second mutation on
the same chromosome can change the disease manifestation from the first
mutation, Ding says.
Mitra explains that the method he
explores as well as the one applied by Cantor and Ding share the novel idea of
co-amplification of two loci on the same molecule. They both carry the message:
"Don't be limited by your amplicon, don't try to PCR amplify both SNPs together,
just co-amplify your SNPs in such a way that if they are from the same DNA
molecule they are going to mingle together . . ." Mitra says. In his view, the
studies show "the only limit you are putting on your distance now is 'can you
keep your DNA intact?'"
Diagnosing the future
According to Cantor, his company believes that "mass spec
has specific diagnostic advantages for nucleic acids over methods that existed
before." Single-molecule haplotypting is one of them, so the company is
wondering if the advantages are sufficient for "an entry into the diagnostic
For clinical applications, molecular
haplotyping offers the potential of accuracy and low cost. For how many clinical
diseases do you need to know the haplotype? "Right now, there are not that
many," Mitra says. The belief is, though, that number will start to explode as
mining of the human genome proceeds.
For more on the
organizations mentioned here, refer to this article at www.genpromag.com
Glimpsing the Future
SNPs are not disease indicators
per se. When specific SNPs are present on the same chromosome, disease
manifestation is not the same as when the SNPs are on different chromosomes. If
haplotyping is to make its way to the clinic, robust, scalable techniques are
needed. According to scientists interviewed for this story, single-molecule
analysis may be a promising approach, particularly co-amplifying two or more
loci on one molecule.LOAD-DATE:
March 05, 2004